PubMed1985
Site-specific binding to human erythrocyte membranes of nitrobenzylthioinosine (NBMPR), un-ionized at physiological pH, was compared with that of hydroxynitrobenzylthioinosine (HNBMPR), pKa 6.4, at graded pH values. Binding of [3H]NBMPR was measured directly, and that of HNBMPR was assayed by competitive inhibition by HNBMPR of [3H]NBMPR binding. Kd and Bmax values for binding of [3H]NBMPR to erythrocyte membranes were independent of pH. Kd values for the competing ligand were determined by mass law analysis of equilibrium binding data using either (a) apparent ligand concentration (dissociated plus undissociated forms of HNBMPR) or (b) the concentration of undissociated HNBMPR. Kd values for HNBMPR calculated with the apparent ligand concentration increased 10-fold as the fraction of HNBMPR molecules present in the dissociated form was increased (by pH changes) from 14 to 88%, whereas Kd values for the undissociated form of HNBMPR were independent of pH. The results presented here demonstrate that the undissociated form of HNBMPR binds more tightly to the transport-inhibitory sites of erythrocytes than NBMPR and suggest that ionization of S6-substituted thiopurine ribonucleosides eliminates or greatly decreases their ability to interact with the binding sites.